Defining process control parameters for cardiac reprogramming PROJECT SUMMARY The prevalence of congestive heart failure (CHF) has risen dramatically in recent years due to improved con- temporary management of ischemic heart disease, the leading cause of death worldwide. The fundamental unresolved issue underlying CHF pathogenesis, however, is irreversible cardiomyocyte (CM) loss. Although various strategies for cardiac repair have been proposed, each approach possesses particular shortcomings, and, in several instances, human translation has proceeded rapidly without adequate mechanistic characteri- zation beforehand. Recently, direct reprogramming of fibroblasts into induced CM-like cells (iCLMs) by GHMT (Gata4, Hand2, Mef2c, and Tbx5) has emerged as a viable, alternative regenerative strategy. Despite highly promising results in preclinical models, however, the efficiency of CM reprogramming remains suboptimal. Therefore, the long-term goal of this research program is to understand the molecular underpinnings of direct cardiac reprogramming as a novel cardiac regenerative and developmental paradigm. The objective of this proposal is to elucidate the essential process control characteristics of cardiac reprogramming. Based on strong preliminary data, our central hypothesis is that specific GHMT protein domains function through precise kinetics to influence the cardiac reprogramming process. Here we outline a comprehensive set of experiments designed to test this hypothesis by pursuing the following two Specific Aims: 1) Determine the biochemical building blocks of cardiac reprogramming and 2) Define the operating parameters for cardiac reprogramming. In Specific Aim #1, we will evaluate the necessity of individual GHMT factors during cardiac reprogramming and assign their function to particular protein domains using a series of full-length, deletion, and mutant con- structs that we have obtained or generated ourselves. In Specific Aim #2, we will systematically determine the ideal order-of-addition and critical temporal windows that are necessary to optimize cardiac reprogramming by applying established and novel reprogramming methodologies. Our approach is innovative because it will uti- lize our robust and validated single-cell assays to interrogate 3 discrete steps during formation of functional iCLMs: genome reorganization, sarcomere assembly, and subtype diversity. This project is significant, there- fore, because it seeks to define the key biochemical inputs and functional design principles that underlie cardi- ac reprogramming. Taken together, the overall impact of this research program is to harness the full potential of cardiac reprogramming as a therapeutic intervention, a system for in vitro disease modeling, and a unique platform for understanding cardiomyogenesis.